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Laboratory Study Design for Risk Assessment of Non-Target Organisms 非靶作用生物风险评估的实验室研究设计

Agricultural Research Service. Laboratory Study Design for Risk Assessment of Non-Target Organisms 非靶作用生物风险评估的实验室研究设计. Rick Hellmich USDA-ARS, Corn Insects & Plant Genetics Research Unit 美国农业部－农业研究服务局，玉米昆虫和植物遗传研究所 Dept. Entomology, Iowa State University 昆虫系，依阿华州立大学. Topics 主题.

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Laboratory Study Design for Risk Assessment of Non-Target Organisms 非靶作用生物风险评估的实验室研究设计

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  1. Agricultural Research Service Laboratory Study Design for Risk Assessment of Non-Target Organisms 非靶作用生物风险评估的实验室研究设计 Rick Hellmich USDA-ARS, Corn Insects & Plant Genetics Research Unit 美国农业部－农业研究服务局，玉米昆虫和植物遗传研究所 Dept. Entomology, Iowa State University 昆虫系，依阿华州立大学

  2. Topics 主题 • Preparing for risk assessment laboratory studies 风险评估的实验室研究准备 • Experimental details 试验细节 • Examples 例子 • Tier 1 study (Univ. NE) 梯度1研究（内布拉斯加州大学） • Tier 2 study (ARS & Univ. Guelph) 梯度1 研究（农业研究服务局和圭尔夫大学 ） • Research and collaboration possibilities 研究和合作的可能性

  3. Preparing for RA Studies 风险评估研究的准备 • Clear purpose and objectives清晰的目的和目标 • From problem formulation 从问题构成开始 • Surrogate concept (case-by-case refinement) 替代概念（案例进化） • Standard list of surrogate species 替代种属的标准清单 • Supplemental species 补充种属 • Species must be laboratory reared种属须实验室培养 • Use of artificial diet is preferable 最好使用人工饲料 • Need to reliably deliver stage specific organisms 需要正确提供特定阶段的生物

  4. Preparing for RA Studies 风险评估研究的准备 • Most protocols are derived from insecticide tests 大部分试验方法都是来自于杀虫剂的试验 • Modified to consider exposure pathway 考虑到暴露途径进行修改 • Bt maize example Bt玉米为例 • Toxin produced in leaf tissues 毒素表达在叶的组织 • Less toxin produced in roots or pollen 在根和花粉毒素很少 • Route of exposure is oral 暴露途径是口

  5. Tiered or Iterative System 分级和迭代系统 • Tier 1: maximum limit dose tests 梯度1：最大限制剂量试验 • > 10X MHD (maximum hazard dose) or HEEE (high-end exposure estimation, 90th percentile) > 10X MHD （最大损害剂量）和 HEEE（高端暴露量估计，90％） • Dose response 剂量反应 • Tier 2: 1X plant material 梯度2： 1X 植物材料 • Refined hazard characterization 精确的损害鉴定 • Higher Tiers: 更高的梯度 • Semi-field and field tests 半田间和田间试验 • Reproduction, life cycle 生殖，生命周期 • Tri-trophic interactions 食物链交换作用 • Triggers for moving to a higher tier should be well defined! 进一步做更高分级试验的阈值需要很好的界定

  6. Experimental Details 试验细节 • Consult relevant compliance standards and guidelines参考相关的标准和指导 • Examples例子 • USEPA 885 Microbial Pesticide Test Guidelines 美国环保局 885 微生物杀虫剂试验指导 • 885.4380 Honey bee testing, Tier I 885.4380 蜜蜂试验，梯度 I • Candolfi, M.P., et al. 2000. Guidelines to Evaluate Side-Effects of Plant Protection Products to Non-Target Arthropods IOBC, BART, EPPO Joint Initiative. IOBC/WPRS (ISBN: 92-9067-129-7) Candolfi, M.P., et al. 2000.评价植保产品对非靶作用节肢动物的副作用。(ISBN: 92-9067-129-7) • Most guidelines based on chemical or microbial insecticides大部分指导都是基于化学和微生物杀虫剂的 • Details for testing GE products often are lacking 测试转基因产品的试验细节还缺少

  7. Bioassays 生物测定法 • Organism – common and scientific names 生物－通俗和科学名 • Stage tested测试的阶段 • Egg, larvae (instar?), adult, multiple generations 卵、幼虫（中间形态？）、成虫、多代 • Test system and conditions测试系统和条件 • Containment, e.g., testing chambers, arenas 容器，如测试皿、测试场 • Temperature, relative humidity, light cycle 温度、相对湿度和光周期 • Food and water delivery 食物和水的提供

  8. Bioassays 生物测定 • Length of test试验时间长短 • Partial or whole stage部分或全部阶段 • Consider longer tests for GE products对于转基因产品考虑更长时间的试验 • Specific measurement endpoints 特定测量指标 • Life-table parameters, e.g., mortality, fecundity 生命表法参数，如死亡率和生殖力 • Other endpoints are possible, e.g., weight, behavior, but risk assessors should agree beforehand how to interpret 其它可能指标，如体重、行为，但是风险评价因子需要事先对怎样解释有共识 • Test material (origin and form)试验材料（来源和型） • Derived frombacteria or plant来源于细菌或植物 • Should be equivalent to form expressed in plant应该于植物中表达的一样 • Dose 剂量 • Consider > 10X MHD or HEEE 考虑 > 10X MHD 或HEEE • Treatment application处理的采用 • Protein in artificial diet, plant material, etc. 蛋白在人工饲料、植物材料中，等。

  9. Quality Control 质量控制 • Assures function of test system 保证试验系统的运行 • Low negative control mortality 低隐性对照死亡率 • Homogeneity of test material 试验材料的同质度 • Stability of insecticidal compound 杀虫药物的稳定性 • Negative control阴性对照 • Carrier/buffer minus test material载体／缓冲液减去试验材料 • Positive control (determine test material is ingested) 阳性对照（确定试验材料被吞噬） • Orally active insecticide口服活性杀虫剂 • Nature of compound (e.g., stomach poison, growth regulator) 试验物的性质（如，胃毒性、生长调控） • Statistics 统计 • Sufficient statistical power (maximize discriminatory ability) 足够统计幂（区分能力最大化） • Power tests, required replicates幂试验，需要重复 • T test, ANOVA, etc. T检验、方差分析， 等 • Good laboratory practices (GLP) recommended 推荐良好实验室质量管理体系 （GLP) • Quality control assures repeatability and interpretability; facilitates transportability of data 质量控制确保重复性和可解释性；有助于数据交流

  10. Higher Tier Testing 更高梯度试验 • Usually involves semi-field or field tests sometimes involves life-cycle or tri-trophic evaluations 通常包括半田间或田间试验，有时包括生命周期或食物链交互作用评价 • Problematic because of complexity and high intrinsic uncertainty; problems of low statistical power 因为很复杂和高内在不确定性，所以会有问题。低的统计学能力也会导致问题。 • Conduct only when tests can reduce uncertainty in risk assessment and only when justified by detection of unacceptable risks at lower tiers of testing 只有当试验能减少风险评价的不确定性和当在低分级试验证实不可接受的风险时才进行更高分级试验

  11. other tests 其它试验 Degradation rates of proteins in soil 蛋白质在土壤中的降解速度 Plant tissue expression data and degradation rates 植物组织表达数据和降解速度 Tier 1 Test Examples 梯度1 试验例子 • acute mammal 急性哺乳动物试验 • rodent 啮齿类 • acute avian 急性禽类试验 • game bird & waterfowl 猎禽和水鸟 • acute fish 急性鱼试验 • cold & warm water spp 冷和温水种属 • acute invertebrate 急性非脊椎动物试验 • daphnia 水蚤 • non-target insects 非靶作用昆虫试验 • honey bee larvae 蜜蜂幼虫 • parasitic hymenoptera 寄生性膜翅目 • green lacewing 草蛉虫 • lady beetle 瓢虫 • Collembola 弹尾目

  12. Dose-Response of Danaus plexippus Larvae to Purified Bt ToxinsDanaus plexippus幼虫对纯Bt毒素的剂量反应关系 Blair D. Siegfried, and Terrence Spencer Department of Entomology 昆虫系 Institute of Agriculture and Natural Resources 农业和自然资源研究所 Nebraska UNIVERSITY OF NEBRASKA-LINCOLN 内布拉斯加州大学

  13. Objectives 目标 • Provide comparative assessment of neonate monarch larval susceptibility to purified Bt toxins 提供可比较的对新生帝王蝶幼虫对纯Bt毒素敏感性的评价 • Determine comparative susceptibility of later instars 测定晚龄的可比较的敏感性

  14. Bioassays 生物测定 • Eggs from field-collected adult females or commercial provider 卵来源于采集田间雌性成虫或供应商 • Neonate (<24 hr) 新生（小于24小时） • Late 2nd (4-day) and Early 4th (7-day) 晚第2（第4天）和早第4（第7天） • Bioassay diet: Multispecies Lepidopteran diet with 2% powdered leaves from common milkweed 生物测定饲料：多种属鳞翅目饲料，另加2％普通乳草叶粉末

  15. Development on Artificial Diet在人工饲料中生长

  16. Bioassays (Cont.) 生物测定（续） • Purified Cry1Ab, Cry1Ac, Cry1F, Cry9C incorporated into diet 纯Cry1Ab, Cry1Ac, Cry1F, Cry9C 加入饲料 • 2 replicates for each toxin; 5 Bt concentrations/rep 每个毒素有2个重复；5个Bt浓度剂量／重复 • Larval weight & mortality recorded at 7 days 7天内记录幼虫体重和死亡率 • Weight determined before and after exposure for later instars (Cry1Ab only) 晚龄（只有Cry1Ab ）在暴露前后测量体重 • Mortality data: probit analysis; 死亡率数据：概率单位分析 Growth Inhibition data: non-linear regression 生长抑制数据：非线性回归分析

  17. Bioassay Methodology 生物测定方法

  18. Results at 7 days7天结果

  19. Sublethal Effects亚致死作用 100 ng/ml Cry 9C Control 对照

  20. Cry1Ab Dose Response Cry1Ab 剂量反应 100 75 Percentage 百分比 Growth Inhibition 生长抑制 50 Mortality 死亡 25 0 0.1 1 10 ng Cry1Ab/mL

  21. Comparative Toxicity (1999-2000) 可比较的毒性（1999－2000） Toxin LC50* EC50 * 毒素 1999 2000 1999 2000 Cry1Ab1.37 4.76 0.13 1.28 Cry1Ac18.54 -- 9.48 -- Cry9C313.9 345.7 25.6 111.2 Cry1F>10,000 >30,000 >10,000 5,200 *ng toxin/ml of diet ng 毒素／ml饲料

  22. Comparative Susceptibility of Larval Instars to Cry1Ab 幼虫对Cry1Ab的可比较敏感性 100 1st Instar 第一龄 2nd Instar 第二龄 75 % Mortality 死亡率 4th Instar 第四龄 50 25 0 1 10 100 Cry1Ab (ng/ml)

  23. Tier 2 Monarch Test梯度 2 帝王蝶试验 Objective: Compare monarch neonates susceptibility to pollen from Bt maize Cry1Ab events MON810, BT11 and 176 deposited on milkweed leaves 目标：比较新生帝王蝶对花粉的敏感性，花粉来自于存积在乳草叶上的有Cry1Ab的 MON810, BT11 和176 Bt玉米

  24. Methods 方法 • Pollen from field-collected plants 田间采集植物的花粉 • Cry1Ab (Events MON810, BT11 and 176) Cry1Ab（来自于MON810, BT11 和176 ） • Pollen sieved with 90 micron screen 用90微米筛子过滤花粉 • Milkweed leaves harvested morning of experiment 乳草叶在试验的早上收获 • Pollen sprayed onto leaf discs 花粉喷洒在叶子上 • Leaf discs put into arena with 2.5% agar 叶子和2.5％胶一起放入试验皿

  25. Methods (cont.) 方法（续） • 1 neonate (< 24 hrs) put into each arena 1只新生幼虫（小于24小时）放入一个试验皿中 • 4 or 5 pollen densities and 0 control tested; 6 replicates per test 4或5花粉密度和0花粉对照；每测试有6个重复 • Larval weight & mortality recorded at 4 days 在4天内记录幼虫体重和死亡率

  26. 170 Mesh Sieve (90 Micron)170网筛（90微米） Siftings 筛屑 Sieved pollen 过筛的花粉

  27. Punch Leaf Disks 打孔叶子

  28. Applying Pollen To Leaf Disks 往叶子上撒花粉

  29. Arena 试验皿

  30. None Bt non-Bt Mean weight (mg) 平均重量（mg） Cry1Ab Event Bt11 Cry1Ab Event Mon810 100- 300 301- 600 601- 1000 1001- 1600 > 1600 Pollen grains per cm2 花粉颗粒每平方厘米

  31. Lab Bioassays - Cry1Ab Event 176实验室生物测定－ Event 176 的Cry1Ab96 Hour Larval Weight 96小时幼虫体重 Hybrids - NK4394 & Max 21 (Bt) 杂交－ NK4394 & Max 21 (Bt) Mean Weight平均体重 (mg) ± 95% CI 100 0 200 400 Pollen per cm2 花粉每平方厘米

  32. Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen帝王碟幼虫对苏云金芽孢杆菌纯蛋白和花粉的敏感性Richard L. Hellmich, Blair D. Siegfried, Mark K. Sears, Diane E. Stanley-Horn, Michael J. Daniels, Heather R. Mattila, Keith G. Bidne, and Leslie C. Lewis PNAS 2001 • Cry1Ab < Cry9C < Cry1F • Older instars less susceptible to Cry1Ab 晚龄幼虫对Cry1Ab更不敏感 • Only event 176 pollen consistently affected larvae 只有event 176 花粉一直影响幼虫

  33. Research & Collaboration Possibilities科研和合作可能性 • There is a need to harmonize lab-based tests for GE products 对转基因产品有必要协调实验室方法 • Tests for standard list of surrogate species 标准清单替代种属的试验 • Identify and develop tests for supplemental species 区别和发展补充种属的试验 • Regionally or ecologically relevant arthropods 地区或生态上来说重要的节肢动物

  34. Summary 摘要 • Surrogate concept and tiered system are important tools for risk assessment 替代概念和分级系统是风险评估很重要的工具 • Higher tier tests should only be conducted 更高分级试验只有当以下情况才进行 • when justified by detection of unacceptable risks at lower tiers of testing 当低分级试验测到不可接受的风险时 • when they can reduce uncertainty in risk assessment 当在风险评估可以减少不确定性时 • Laboratory tests 实验室试验 • Well defined 很好地定义 • Quality control facilitates repeatability of tests and transferability of data 质量控制有助于试验的可重复性和数据交流 • Many opportunities for research and collaboration 科研和合作的很多机会

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